Endotoxin, also known as lipopolysaccharide (LPS), is an integral component of the Gram-negative bacterial cell membrane and is responsible for many, if not all, of the toxic effects that occur during Gram-negative bacterial sepsis.
LPS from Gram-negative bacteria induces the amoebocytes of horseshoe crabs to aggregate and to degranulate. Presumably, the LPS-induced coagulation cascade represents an important defense mechanism used by horseshoe crabs against invasion by Gram-negative bacteria. The amoebocyte lysate constituted as the Limulus amoebocyte lysate (LAL) test has been used for decades as a tool for detecting trace concentrations of endotoxin (LPS) in solution. The molecular mechanism of coagulation in horseshoe crab has been established and it involves a protease cascade. This cascade is based on 3 kinds of serine protease zymogens, Factor C, Factor B, proclotting enzyme, and one clottable protein, coagulogen. Being the initial activator of the clotting cascade, Factor C functions as a biosensor that responds to LPS. Once Factor C is “activated” by LPS, the active moiety created has the ability to activate Factor B and to hydrolyse synthetic tripeptide substrates.
Factor C activity is the basis of a very sensitive assay for femtogram levels of endotoxin used in the quality control of pharmaceutical products and the like. The importance of Factor C in the detection of endotoxin has thus led to the expression of recombinant Factor C (rFC) as an alternative source that should alleviate the recognized drawbacks with conventional amoebocyte lysate like seasonal variation in the sensitivity of detection of endotoxin.
For endotoxin specific assays, Factor C protein has been purified and cloned. Upon activation by LPS, recombinant Factor C acts on a synthetic substrate present in the assay mixture to generate a detectable signal, thereby indicating the presence of LPS in a given sample. In particular, a fluorogenic substrate produces a fluorescent signal in proportion of the endotoxin concentration in the sample. Factor C protein has been purified and cloned for its application in endotoxin-specific assays.
Nakamura et al. (1986, Eur. J. Biochem. 154:511-521) describe the isolation and characterization of native Factor C protein from the horseshoe crab Tachypleus tridentatus. The cDNA sequence encoding Factor C protein from T. tridentatus was published by Muta et al. (1991, J. Biol. Chem. 266:6554-6561). The cDNA sequence encoding Factor C protein from the horseshoe crab Carcinoscorpius rotundicauda was published by Ding et al. (1995, Molecular Marine Biology and Biotechnology 4:90-103).
The recombinant expression of Factor C from C. rotundicauda in E. coli was described in Roopashree et al. (1995, Biochem. Mol. Biol. Intl. 35:841-849). Here, the expression of a 108 kDa proenzyme and the activated forms of 78 kDa and 52 kDa was shown by immunodetection.
The recombinant expression of Factor C protein from C. rotundicauda in the yeast Pichia pastoris is described in Ding et al. (1996, U.S. Pat. No. 5,858,706). The recombinant expression of Factor C protein from C. rotundicauda in Saccharomyces cerevisiae is described in Dwarakanath et al. (1997, Biotechnology Letters 19:1147-1150).
The recombinant expression of Factor C protein from C. rotundicauda in mammalian COS-1 cells is described in Dwarakanath et al. (1997, Biotechnology Letters 19:357-361). Here, Factor C protein was expressed and protein bands with a molecular weight of 132 kDa, 130 kDa and 63 kDa were detected. The proteins were not secreted, not soluble, and not active, but were rather insoluble, associated with the cell debris fraction.
The recombinant expression of Factor C protein from C. rotundicauda in insect cells (stable transfected Sf9 cells) is described in Wang et al. (2001, Biotechnology Letters 23:71-76). Here, Factor C protein was secreted into the supernatant with a molecular weight of 132 kDa, which indicated glycosylation of the protein. The Factor C obtained was functionally active in the sense that it could bind endotoxin (LPS). However, no conversion into an enzymatically active protease was shown. The recombinant expression of Factor C protein from C. rotundicauda in insect cells is also described in Wang et al. (2002, J. Biol. Chem. 277:36363-736372). Here, Factor C was cloned and transfected into Drosophila S2 cells and was expressed as a glycosylated soluble protein, which was secreted into the culture supernatant. The recombinant Factor C protein was capable of binding LPS, but was not cleaved to become an enzymatically active protease. The recombinant expression of Factor C protein from C. rotundicauda in insect cells is furthermore described in U.S. Pat. No. 6,645,724 using the baculovirus system for expression in Sf9 cells.
Factor C protein is a complex eukaryotic protein, which requires several conversion steps and secondary modifications to become an active protease. The recombinant expression in prokaryotes (e.g., E. coli) does not provide glycosylation, cleavage into H-chain and L-chain and correct disulfide bond formation. The cytosolic expression in simple eukaryotic expression systems (e.g., yeast) provided Factor C, which was capable to bind LPS, but which was not activated upon LPS binding, i.e., there was no conversion from the zymogen form into an active protease. When using yeast cells (Pichia or Saccharomyces) for expression, it was not possible to obtain recombinant Factor C as secreted protein. The expression in a mammalian cell line did also not provide active secreted protein. Furthermore, the expression in stable transformed insect cells provided secreted protein, which was capable to bind LPS. However, the activation by LPS was not shown in this system. Finally, the expression in insect cells using a baculovirus expression system provided secreted Factor C protein, which was capable to bind LPS, and which was converted into an active serine protease zymogen upon LPS binding.
From all experience so far gained it was concluded by the experts succeeding with the expression of active Factor C protein in insect cells, that “expression in insect cells rather than in a prokaryotic or simple eukaryotic expression system is suitable for producing rFC with full biological activity. Furthermore, horseshoe crabs and insects belong to the same phylum, Arthropoda, and so insect cells might more closely resemble the cells of the horseshoe crab than yeast cells in their physiology and biochemistry. Thus, rFC produced in insect cells might more closely resemble the protein as purified from the horseshoe crab and retain the bioactivity of having a serine protease activity activated by LPS” (see WO 99/15676 on page 2, “Summary of the Invention”).
Since that time, i.e., more than 13 years after the publication of WO 99/15676, no further attempts have been made with respect to the recombinant production of active Factor C protein. Obviously, in view of the results obtained over the years from recombinant expression in various host systems, and in view of the unequivocal assessment given by the top experts in the field in WO 99/15676, the baculovirus expression system in insect host cells was considered as the gold standard for recombinant production of active Factor C protein.